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Biomedical subjects

H Sugawa

Publications and source records attributed to H Sugawa.

At least 19 recordsLinked to original sources
PubMed2001

Improved hepatic microcirculation by human soluble urinary thrombomodulin in the xeno-perfused porcine liver.

PURPOSE: Both the protein C/thrombomodulin system and the heparin/anti-thrombin III system are major physiological anticoagulant systems, which may also play a major role in preserving the hepatic microcirculation in xenogeneic liver transplantation. To compensate for the functional incompatibilities of the porcine thrombomodulin (TM)-cofactor activity beyond species for human thrombin, soluble human TM protein was tested in xenogeneic perfusion of the porcine liver. MATERIALS AND METHODS: The livers were harvested from adult female pigs and perfused through the portal vein (PV) and hepatic artery (HA) for 2 hr, with fresh human blood in group 1 (n=5), fresh porcine blood (10 units/ml) in group 2 (n=5), and fresh human blood with TM (50,000 units/1.5 l) in group 3 (n=5). The tissue PO2 level, tissue blood flow, PV and HA pressures were all continuously monitored. Circulating perfusate and liver tissue samples were periodically obtained for blood chemistry and histologic analyses. RESULTS: The activated protein C (aPC) level was significantly elevated in the TM-treated group 3 (47.5%+/-3.5% at preperfusion and 51%+/-2.8% after 120 min of perfusion) in comparison to group 1 (32.3%+/-7.2% and 35.3+/-12.0%). The hepatocyte enzyme release of aspartate aminotransferase (AST) was suppressed significantly more in group 3 (238.2+/-107 IU/l), than in group 1 (672.3+/-160 IU/l) at 2 hr after reperfusion. In group 3, the tissue PO2 levels and tissue blood flow also remained significantly higher throughout the perfusion. The platelet counts in the perfusate remained significantly higher in group 3 (37.1% to 74.3% of the preperfusion level) than in group 1 (4.4% to 14.7%), after 0 to 80 min of perfusion. According to the histologic findings, the degree of interlobular hemorrhaging and congestion decreased remarkably more in group 3 than in group 1. CONCLUSION: These findings thus indicated that soluble thrombomodulin protein extracted from human urine remarkably improved hepatic microcirculation in the xenoperfused porcine liver. The thrombomodulin/protein C system might, thus, play an important role in restoring the physiological anticoagulant system in the xenoperfused porcine liver.

Animals↗
PubMed2000

A novel activating mutation in the thyrotropin receptor gene in an autonomously functioning thyroid nodule developed by a Japanese patient.

OBJECTIVE: A number of activating mutations of the thyrotropin receptor (TSHR) have been found in autonomously functioning thyroid nodules (AFTNs) in European patients. We aimed to study TSHR mutation in AFTNs in Japanese patients because no TSHR activating mutation has been found by previous incomplete studies. DESIGN: A typical AFTN developed in a 69-year-old Japanese woman was studied. METHODS: The entire exon 10 of the TSHR cDNA was sequenced. Functional studies were done by site-directed mutagenesis and transfection of a mutant construct into COS-7 cells. RESULTS: We identified a novel heterozygous TSHR gene mutation, Leu512-->Arg (L512R; CTG-->CCG), from the AFTN. The mutation was not detected in the adjacent normal thyroid tissue. COS-7 cells transfected with L512R mutant TSHR expression vector exhibited a 3.3-fold increase in basal cAMP level compared with that of cells transfected with wild-type TSHR DNA, confirming that the mutation was the direct cause of the AFTN. TSHR activating mutations involving the third transmembrane helix reported to date are S505R/N and V509A as well as L512R. An in vitro site-directed mutagenesis study encompassing residues 505-513 revealed that mutations involving residues other than these three did not show constitutive activation. CONCLUSION: This is the first TSHR activating mutation found in a Japanese patient, although true prevalence of TSHR activating mutations in AFTNs developed in Japanese patients remains to be elucidated. In addition, functional studies suggested that amino acid residues in the third transmembrane helix maintaining inactive conformation of the TSHR seem to be located on the same surface of the alpha-helix, possibly making interhelical bonds with another helix.

Aged↗
PubMed1999

Analysis of Fas system in pulmonary injury of graft-versus-host disease after rat intestinal transplantation.

The lung is one of the primary targets of acute graft-versus-host disease (GVHD), which is the principal complication that occurs after allogeneic intestinal transplantation. The purpose of this study is to investigate the involvement of Fas/Fas ligand system in pulmonary injury after rat semi-allogeneic intestinal transplantation. The lungs were serially harvested from LEW x BN F1(LBNF1) recipients of either LEW heterotopic intestinal allografts or LBNF1 isografts, on days 1, 3, 5, 9, and 13 posttransplant. In light microscopy, pulmonary injury became apparent on day 13 in the allogeneic combination, showing a thickening of the alveolar septa. The incidence of apoptosis, examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) biotin nick end-labeling, was observed to increase steadily in the alveolar cells accompanied by a progression of GVHD. In an immunohistochemical study, Fas was constitutively expressed in the lung, although Fas ligand was expressed most extensively on day 9. The immunoreactivity of both Fas and Fas ligand were observed in alveolar cells, in addition to leukocytes. An analysis by reverse transcription polymerase chain reaction also revealed that the expression of Fas mRNA was constitutive without any significant change, although that of Fas ligand mRNA increased substantially and peaked on day 9, which was significant compared to the isogeneic combination. In conclusion, transcriptionally up-regulated Fas ligand and increased number of apoptosis suggests that the Fas system may play a role in the pathophysiology of GVHD-induced pulmonary injury.

Animals↗
PubMed1999

Stable gene expression with VSV-G pseudotyped-retrovirus vector in the rat liver.

BACKGROUND: Pseudotyped-retrovirus-mediated gene transfer to the regenerating rat liver was investigated in vivo and the findings were compared with those for retrovirus-mediated gene transfer. MATERIALS AND METHODS: Four weeks prior to gene transfer, the spleen was transpositioned to the left subcutaneous position to develop a port-splenic shunt. Twenty-four hours after a partial hepatectomy (68%) was performed, the liver was perfused in situ and kept in contact with either a pseudotyped-retrovirus vector encoding LacZ (7 x 10(7) cfu/ml, Group 1) or a retrovirus vector encoding LacZ (1 x 10(4) cfu/ml, Group 2) for 30 min. The animals were sacrificed at various points after gene transfer, and X-gal staining, reversed polymerase chain reaction (RT-PCR), and ONPG assay were performed to detect the transferred LacZ cDNA. RESULTS: In X-gal staining, the transferred LacZ cDNA started to show a strong beta-galactosidase activity in 30 to 50% of the hepatocytes at 3 days after gene transfer. Positive staining continued to be recognized until 28 days with a slight decrease in its intensity thereafter. On the other hand, Group 2 animals showed weak staining, which was observed in about 10 to 15% of the hepatocytes from 3 days after gene transfer and then decreased thereafter. In RT-PCR, positive mRNA of LacZ was detected constitutively until 28 days after gene transfer in Group 1, whereas two-thirds of the samples showed a negative band in Groups 2 at 3 days after gene transfer. CONCLUSION: In conclusion, the pseudotyped-retrovirus vector was useful in establishing a stable and strong expression of the in vivo gene transfer, while targeting the regenerating liver.

Animals↗
PubMed1999

Analysis of the Fas system and Bcl-2 in rat liver allograft rejection.

BACKGROUND: Apoptosis is involved in the mechanism of cell death observed in liver allograft rejection. The liver cells are sensitive to Fas-mediated apoptosis; however, little is known about the involvement of the Fas system in liver allograft rejection. We used rat models to investigate the expression of Fas/Fas ligand and apoptosis-related proteins during liver allograft rejection. MATERIALS AND METHODS: DA rats to Lewis, and Lewis to Lewis orthotopic liver transplantation were performed; liver samples were collected on days 1, 3, 5, 7, and 9 postoperatively (each n = 3). Apoptosis was monitored by TUNEL and electron microscopy. The expression of Fas, FasL, bcl-2, and bax was examined at the mRNA level and by means of immunohistochemistry. RESULTS: The TUNEL index in the allografts and isografts on day 7 was 20.1 +/- 1.5 and 7.7 +/- 2.6/1000 cells, respectively. Fas and bax mRNA were constitutively expressed in both of the groups. The expression of Fas ligand mRNA in the allografts which rose on day 5 was 10 times stronger compared to that in the isografts. On the other hand, bcl-2 mRNA was generally expressed in the isografts while it decreased in the allografts. The immunohistochemical analysis also showed an increased reactivity of Fas ligand on day 5 in the allograft, which was observed both in parenchymal and nonparenchymal cells. CONCLUSIONS: These results strongly suggest that Fas/Fas ligand interaction mediates the liver injury during allograft rejection. In addition, other regulatory factors of apoptosis, such as bcl-2, might also be involved in this pathogenesis.

Animals↗
PubMed1999

Expansion of helper T-cell recognition in mice immunized with a synthetic peptide corresponding to the C-terminal thyrotropin receptor-specific insert.

We previously reported that immunization with a synthetic peptide of human thyrotropin receptor (TSH-R) expanded humoral autoimmunity to TSH-R (Sugawa H, Ueda Y, Ueda M, Kosugi S, Ichiyama S, Mori T. Immunization with the 'immunogenic Peptide' of TSH receptor induces oligoclonal antibodies with various biological activities. Peptide 1998;19:1303-7.). In the present study, we examined this phenomenon at the T-cell level. Balb/c mice were immunized with a synthetic peptide corresponding to the C-terminal-specific insert of human TSH-R. Spleen cells were collected and subjected to antigen-specific ELISPOT assay. The number of interleukin 4-secreting cells specific to P354-367 increased within 3 weeks. Cells responding to the other peptides increased 7 weeks after immunization. This phenomenon was not observed in mice immunized with bovine serum albumin alone. During immunization, numbers of interferon-gamma-secreting lymphocytes were not changed significantly. These results indicated that immunization with C-terminal TSH-R-specific insert peptide causes fluctuation in the type 2 helper T-cell population but not type 1 Th cells against the TSH-R, and the recognition repertoire of type 2 helper T cell was expanded by the peptide.

Animals↗
PubMed1998

Differences in the electrophysiological response to I- and the inhibitory anions SCN- and ClO-4, studied in FRTL-5 cells.

The electrophysiological properties of the Na+/I- symporter (NIS) were examined in a cloned rat thyroid cell line (FRTL-5) using the whole-cell patch-clamp technique. When the holding potential was between -40 mV and -80 mV, 1 mM NaI and NaSCN induced an immediate inward current which was greater with SCN- than with I-. The reversal potential for I- and SCN- induced membrane currents was +50 mV. This is close to the value of +55 mV calculated by the Nernst equation for Na+. These results are consistent with I- and SCN- translocation via the NIS that is energized by the electrochemical gradient of Na+ and coupled to the transport of two or more Na+. There was no change in the membrane current recording with ClO-4 indicating that ClO-4 was either not transported into the cell, or the translocation was electroneutral. ClO-4 addition, however, did reverse the inward currents induced by I- or SCN-. These effects of I-, SCN- and ClO-4 on membrane currents reflect endogenous NIS activity since the responses duplicated those seen in CHO cells transfected with NIS. There were additional currents elicited by SCN- in FRTL-5 cells under certain conditions. For example at holding potentials of 0 and +30 mV, 1 mM SCN- produced an increasingly greater outward current. This outward current was transient. In addition, when SCN- was washed off the cells a transient inward current was detected. Unlike SCN-, 1-10 mM I- had no observable effect on the membrane current at holding potentials of 0 and +30 mV. The results indicate FRTL-5 cells may have a specific SCN- translocation system in addition to the SCN- translocation by the I- porter. Differences demonstrated in current response may explain some of the complicated influx and efflux properties of I-, SCN- and ClO-4 in thyroid cells.

Animals↗
PubMed1998

Successful gene transfer to the porcine liver in vivo with an adenoviral vector.

In vivo gene transfer to the porcine liver was tested with adenoviral vector to achieve molecular biological graft modulation. In adult female pigs immunosuppressed with cyclophosphamide, cyclosporine, and prednisolone, the liver was surgically isolated and flushed out with cold lactate Ringer solution (4 degrees C), by means of a pump-controlled bypass of the portal vein and the inferior vena cava in Groups A and D. In Group A (n = 4), 2 x 1011 pfu of the adenoviral vectors (pAdexCALacZ) were injected through the left hepatic artery during cold ischemia. In Group (n = 4), 2 x 1011 pfu of adenovirus vectors were injected through the auricular vein in a one-shot manner without a laparotomy. In Group C (n = 4), 2 x 1011 pfu/ml of adenoviral vectors were injected through the hepatic artery in a one-shot manner, without a surgical isolation of the liver. Group D (n = 4) animals received the same protocol as Group A except for the fact that they did not receive the immunosuppressive regimen. In a polymerase chain reaction, a transfected LacZ sequence was detected until POD 28 in Group A, but not in the other groups. In 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining, only the Group A animals revealed apparent staining predominantly in the portal area at POD 2, which then continued to be recognized until POD 28. The in situ perfusion of the liver combined with immunosuppression is thought to provide an ideal environment for the liver-directed adenovirus-mediated gene transfer to the porcine liver, by enabling a long contact with a high titer of the adenoviral vector.

Adenoviridae↗
PubMed1998

Immunization with the "immunogenic peptide" of TSH receptor induces oligoclonal antibodies with various biological activities.

Balb/c mice were immunized with a synthetic peptide (P354-14) corresponding to "the immunogenic peptide" of human thyrotropin receptor (hTSH-R). Through screening for binding to the peptide, we obtained several monoclonal antibodies with various biological activities: thyroid stimulation (SAb), inhibition of TSH stimulation (BAb) and no significant effect on cAMP production. One of the stimulatory clones was further studied. This clone enhanced cAMP production in Cos-7 cells transformed with the truncated TSH-R cDNA deleting the immunogenic peptide. These results indicated that the immunogenic peptide of the TSH-R induces oligoclonal anti-TSH-R antibodies, although the region is not essential for the functional epitope.

Animals↗
PubMed1998

Incidence and characteristics of thyroid dysfunction following interferon therapy in patients with chronic hepatitis C.

Thyroid functions were analyzed before, during and after interferon (IFN) therapy in patients with chronic hepatitis C. According to the results of routine thyroid function tests and measurements of the levels of anti-thyroid autoantibody prior to the therapy, patients were divided into 2 groups; Group A (19 patients) had at least one abnormal finding related to the thyroid, and Group B (40 patients) did not show any abnormality. Five patients (26%) in Group A and 4 (10%) in Group B showed thyroid dysfunctions which were very clearly reflected by thyrotropin (TSH) measurements. Interestingly, the time of peak TSH elevation in Group A (mean +/- SD, 4.3 +/- 0.8 months) was significantly earlier than that in Group B (6.8 +/- 0.8). Most patients in Group B were diagnosed as having destructive thyroiditis. These findings may suggest that the pathogenesis of IFN-induced thyroid dysfunction consists not only of exacerbation of pre-existing thyroid autoimmunity but also of de novo destructive changes even in the intact thyroid before IFN therapy.

Adult↗
PubMed1997

Aspartate-474 in the first exoplasmic loop of the thyrotropin receptor is crucial for receptor activation.

Asp-474 in the first exoplasmic loop of the thyrotropin receptor (TSHR), which is conserved among all glycoprotein hormone receptors, was mutated to Glu which is similarly charged but is longer by one methylene group and expressed in Cos-7 cells. Cells expressing this mutant receptor showed markedly impaired TSH- and TSAb (thyroid stimulating antibody)-stimulated cAMP responses with no effect on TSH binding affinity when compared with cells expressing a similar number of wild-type receptors. These results suggest the importance of Asp-474 in TSHR in receptor activation as demonstrated for LHR (lutropin receptor), but this, unlike LHR, is not due to the electrostatic interaction of this Asp residue with the alpha-subunit Lys-91 of the hormone.

Animals↗
PubMed1997

Recent trends in the management of Graves' hyperthyroidism in Japan: opinion survey results, especially on the combination therapy of antithyroid drug and thyroid hormone.

An opinion survey concerning the management of Graves' hyperthyroidism was conducted among the council members of the Japan Thyroid Association. The selection of 3 major treatments by 90 respondents for their patients was 98.6 +/- 4.2% for antithyroid drug (ATD), 7.8 +/- 12.6% for partial thyroidectomy and 5.2 +/- 8.1% for radioiodide. They expressed a movement away from the past trend of surgery because of postoperative complications and unsatisfactory therapeutic results, and they assumed a further reduction in the future. On the other hand, the frequency of radioiodide treatment was not considered to have decreased greatly, and they expected a slight increase in the future. Of the respondents, 65% suggested that hyperthyroidism should be completely cured even if the patient would fall into hypothyroidism. The major reasons for choosing surgery or radioiodide after ATD were the adverse effects of ATD and the age and social backgrounds of the patients. Large goiter size was the 3rd reason for surgery but was a minimal indicator for radioiodide. As for ATD treatment, none of the respondents reported the routine application of any uniform fixed-time therapy protocol. Japanese Graves' patients were shown to be less responsive to ATD than Caucasian patients. This was assumed to result at least from high iodide intake, and half of them had ordered their patients to restrict iodide intake. Furthermore, 78% of them had treated with a combined therapy of ATD and thyroid hormone. Most of them apply this for selected patients mainly to lower TSH receptor antibody activity, to better control their patients and to reduce the goiter size. All but 8 (9%) did not give T4 (or T3) after the cessation of ATD, and they felt this to be unnecessary, doubtful about the effect, unsuitable or even possible to induce recurrence. The excellent findings reported by Hashizume et al. (N Engl J Med 324: 947-953, 1991) are well known among them. However, most of them did not agree with the efficacy of the protocol to reduce TRAb or to improve the remission rate, and 90% of the respondents did not intend to apply the protocol immediately. In conclusion, the Japanese thyroidologists were shown to highly prefer ATD, and they intended to treat their patients for longer periods of time only by ATD until clinical remission is achieved. The combination therapy is widely used, but most of them do not consider it effective. The therapeutic protocol reported by Hashizume et al. was not accepted widely in Japan.

Antithyroid Agents↗
PubMed1997

Effectiveness of a short-term steroid treatment on the reduction in goiter size in antithyroid drug-treated patients with Graves' disease.

Reduction of goiter size is one of the criteria necessary to determine clinical remission in antithyroid drug (ATD)-treated Graves' patients. To facilitate goiter reduction or to achieve quick remission, a short-term steroid treatment was administered to 5 Graves' patients. These patients had been treated with ATD for a considerable period of time, had maintained euthyroidism with negative or weak thyrotropin-binding inhibitor immunoglobulin (TBII), but still had an enlarged goiter and remained T3 unsuppressive. Betamethazone was initially given 1.5 mg daily and then gradually tapered to 0 mg for 3 months. Compared to the 6-month observation period or the pre-medication period, goiter reduction exceeding 0.7 cm was achieved in all 5 patients during the steroid treatment. These reductions partly reversed to an extent smaller than pre-treatment levels in 2 patients, but continuous goiter reductions were observed for at least 3 months after steroid cessation in 3 patients. In 2 of these reduced goiter patients, T3 suppressibility was confirmed, and they were diagnosed in remission. During steroid administration, serum T3 and TSH concentrations were lowered but reversed shortly, serum fT4 concentrations did not change, and TBII levels became negative in all patients including the 2 with weak positive values before the treatment. In conclusion, a short-term steroid treatment for goitrous ATD-treated Graves' patients appears promising in achieving goiter reduction or remission.

Adult↗
PubMed1996

Establishment and characterization of a Chinese hamster ovary cell line, CHO-4J, stably expressing a number of Na+/I- symporters.

The cDNA of the Na+/I- symporter playing a key role in thyroid iodide transport was cloned very recently. To characterize its function, we transfected the Na+/I- symporter gene into Chinese hamster ovary (CHO) cells and established a cell line stably expressing a number of rat Na+/I- symporters, named CHO-4J. Iodide uptake was dependent on [Na+] and reached a plateau within 30 min. Kinetic studies revealed that the K(m) for iodide was 35 microM, similar to that of FRTL-5 thyroid cells. The maximal velocity (Vmax) at cell protein level was 6-10-fold higher than in FRTL-5 cells and that at single cell level was approximately 1000-fold higher. CIO4- and SCN- dose-dependently inhibited iodide uptake in a competitive manner. The Ki was 1.5 and 16 microM, respectively. Iodide efflux from CHO-4J cells was apparently slower (t1/2 = 15 min) than FRTL-5 cells (t/2 = 2 min). Electrophysiological characteristics were examined using the whole cell patch clamp technique. Rapid inward current was observed when CHO-4J cells were perfused with 50-1000 microM Nal, suggesting a transport stoichiometry of at least 2 Na+ per I-. The current-voltage relation revealed that this current was membrane potential-dependent. The reversal potential was very close to that of Na+ in agreement with dependency on the Na+ electrochemical gradient. CHO-4J cells with a slow iodide efflux, expressing a number of Na+/I-symporters whose characteristics are identical to those of FRTL-5 cells will function as a new tool for sensitive of iodide uptake.

Animals↗
PubMed1996

Laboratory tests necessary at the first examination of patients with suspected thyroid disorders.

In order to determine what and how many test items are necessary for the initial tests when examining patients with thyroid disorders, a questionnaire was sent to the council members of the Japan Thyroid Association. Thyroid disorders were divided into 4 categories; hyperthyroidism, hypothyroidism, diffuse goiter, and nodular goiter. The questionnaire was designed to assess: 1) tests routinely ordered at the initial examination; 2) minimum test battery necessary; 3) the 5 most important tests in order; and 4) selection of only 2 tests at most. The data on 112 completed questionnaires were collected and analyzed. When the choice was limited to only 2 tests, TSH and fT4 were commonly selected for hyperthyroidism hypothyroidism and diffuse goiter, but these 2 tests would be insufficient for the differentiation of disorders. In the case of nodular goiter, ultrasonogram and fine needle aspiration biopsy (FNA) were selected. The mean number of routinely ordered tests was 6.62 +/- 2.00 (S.D.) for hypothyroidism and 8.06 +/- 2.48 for nodular goiter, but many fewer tests were cited as absolutely necessary. In hyperthyroidism, 4.56 +/- 1.92 tests were required, in hypothyroidism 4.39 +/- 1.92, in diffuse goiter 4.93 +/- 1.75, and in nodular goiter 5.15 +/- 2.13. Most of the function-related tests, ultrasonogram and autoantibodies were commonly selected, while TBII and thyroid 123I or 99mTcO4 uptake in hyperthyroidism, and FNA, Tg and scintigram in nodular goiter were considered to be for specific purposes. Summarizing all these, the authors propose 5 or 6 tests to be necessary as the initial tests when examining patients with 4 individual thyroid disorders.

Autoantibodies↗